Showing papers on "Protein primary structure published in 2001"
TL;DR: The sequence- Specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.
626 citations
TL;DR: Structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.
Abstract: In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLphi or YXXphi endocytosis signals (phi is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.
463 citations
TL;DR: The purification of this protein complex from Arabidopsis showed the presence of further forms of TOM20, which comprises a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria.
Abstract: The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.
190 citations
TL;DR: It is significant that β‐defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the β‐ defensin‐specific fold appears to accommodate a wide range of different amino acids at most sequence positions.
Abstract: Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian β-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human β-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that β-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the β-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because β-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.
160 citations
TL;DR: The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum and it is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6.
117 citations
TL;DR: The hypothesis that LeuA13 and LeuB17 form part of a second receptor-binding site in the insulin molecule finds some support in terms of their conservation during vertebrate evolution, although the site is probably absent in some hystricomorph insulins.
112 citations
TL;DR: The cloning and sequencing of the human p8 cDNA showed the presence of a bipartite motif of nuclear targeting, indicating that p8 may function within the nucleus and this presumption has been confirmed by transfection of COS-7 cells with the p7 cDNA followed by immunostaining with specific antibodies.
Abstract: We have previously identified a new rat mRNA, provisionally named p8, which showed a strong, but transient, induction in the pancreas in response to acute pancreatitis. We report here the cloning and sequencing of the human p8 cDNA. The human p8 polypeptide is 82 aminoacids long and shows an overall identity of 74% with the rat counterpart. The complete structure of the p8gene was also established. The p8 gene comprises three exons separated by two introns and the gene was mapped to chromosome 16. Analysis of the p8 primary structure suggested the presence of a bipartite motif of nuclear targeting, indicating that p8 may function within the nucleus. This presumption has been confirmed by transfection of COS-7 cells with the p8 cDNA followed by immunostaining with specific antibodies. p8 mRNA expression is induced in some, but not all, cells by serum starvation and ceramide. To analyze the p8 function, we stably transfected HeLa cells with a p8 expression plasmid, and measured their growth and their sensitivity to apoptosis. Response to TNFα and staurosporine-induced apoptosis was not modified by p8 expression. However, cells expressing p8 grew significantly more rapidly.
104 citations
TL;DR: Computational results provide some new insights into the possible role and importance of PP13 in various processes of the human body and can be of help in the initial steps of further functional research.
Abstract: The primary structure of the newly sequence analysed placental tissue protein 13 (PP13) was highly homologous to several members of the beta-galactoside-binding S-type lectin (galectin) family. By homology modelling, the three-dimensional structure of PP13 was built based on high-resolution crystal structures of homologues and also their characteristic 'jellyroll' fold was found in the case of PP13. Our model has been deposited in the Brookhaven Protein Data Bank. By multiple sequence alignment and structure-based secondary structure prediction, we underlined the structural similarity of PP13 with its homologues. The secondary structure of PP13 was identical with 'proto-type' galectins consisting of a five- and a six-stranded beta-sheet, joined by two alpha-helices, and galectins' highly conserved carbohydrate-recognition domain (CRD) was also present in PP13. Of the eight consensus residues in the CRD, four identical and three conservatively substituted were shared by PP13. By docking simulations PP13 possessed sugar-binding activity with highest affinity to N-acetyllactosamine and lactose typical of most galectins. All ligands were docked into the putative CRD of PP13. Based on several lines of evidence discussed in this paper demonstrating that PP13 is a novel galectin, PP13 was also designated galectin-13. These computational results provide some new insights into the possible role and importance of PP13 in various processes of the human body and can be of help in the initial steps of further functional research.
99 citations
TL;DR: Observations suggest that the regular arrangements of COO− groups in the D domain side chains may be important for specific control of crystal growth.
Abstract: The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa. The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp. Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that the regular arrangements of COO− groups in the D domain side chains may be important for specific control of crystal growth.
92 citations
TL;DR: Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation, including the interaction with a variety of surface metals on the substratum.
Abstract: Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20 kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902 bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357 Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed.
91 citations
TL;DR: Comparative reverse transcription-PCR and Northern-blot analyses detected PAD type I mRNA only in the epidermis and uterus, providing evidence that its expression is under the control of the sex steroid hormone.
Abstract: Peptidylarginine deiminases (PADs), a group of post-translational enzymes, catalyze the conversion of protein-bound arginine residues to citrulline residues in a calcium ion-dependent manner and are widely distributed in various organs of vertebrates Although the existence of four isoforms of PAD (types I, II, III, and IV) is reported in rodents, the relative functions of the isoforms with respect to their colocation in the tissues have yet to be explored In this study, we cloned the full-length cDNA encoding mouse PAD type I by screening a uterine cDNA library and using the RACE method This cDNA consists of an open reading frame of 1989 bases encoding 662 amino acids (73 823 Da), a 5′-untranslated region of 127 bases and a 3′-untranslated region of 1639 bases Comparative reverse transcription-PCR and Northern-blot analyses detected PAD type I mRNA only in the epidermis and uterus Administration of estrogen to adult ovariectomized mice increased the content of PAD type I mRNA in the uterus, providing evidence that its expression is under the control of the sex steroid hormone We also cloned the full-length cDNAs of mouse PAD type III and type IV by the reverse transcription-PCR and RACE methods The primary structure of PAD type III contains 664 amino acids (75 098 Da) deduced from the coding region of 1995 bases, and the primary structure of PAD type IV consists of 666 amino acids (74 475 Da) deduced from the coding region of 2001 bases Comparison of the deduced amino acid sequences of all four isoforms of PAD showed about 50% identity with each other, the 3′ regions being highly homologous compared with the 5′ regions
TL;DR: The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBankTM/EBI data banks showed that PcF shared no significant homology with other known sequences.
Patent•
29 Jun 2001
TL;DR: Glycosylated polypeptides comprising the primary structure NH2-Px-X-Py-COOH, wherein Px is an Nterminal part of a poly peptide Pp of interest, Py is a C-terminal parts of said poly peptile Pp, and X is a peptide addition comprising or contributing to a glycosylation site.
Abstract: Glycosylated polypeptides comprising the primary structure NH2-X-Pp-COOH wherein X is a peptide addition comprising or contributing to a glycosylation site, and Pp is a polypeptide of interest or comprising the primary structure NH2-Px-X-Py-COOH, wherein Px is an N-terminal part of a polypeptide Pp of interest, Py is a C-terminal part of said polypeptide Pp, and X is a peptide addition comprising or contributing to a glycosylation site The glycosylated polypeptides having improved properties as compared to the polypeptide of interest
TL;DR: The primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine is determined, which could provide nutritionally important amino acid nitrogen to ruminants.
Abstract: We determined the primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. Ovine PepT1 (oPepT1) cDNA was 2829-bp long, encoding a protein of 707 amino acid residues with an estimated molecular size of 78 kDa and an isoelectric point (pI) of 6.57. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. The transport process was electrogenic and pH dependent, but independent of Na + , Gland Ca 2+ . The oPepT1 displayed a broad substrate specificity for transport of neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (K t ) ranging from 27 μmol/L to 3.0 mmol/L. No responses were detected from tetrapeptides or free amino acids. Northern blot analysis demonstrated that oPepT1 was expressed in the small intestine, omasum and rumen, but was not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide nutritionally important amino acid nitrogen to ruminants.
TL;DR: The results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.
Abstract: An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities). The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.
TL;DR: Results indicate that, of the two isoforms, DPPX-S is preferentially expressed in the brain tissue as the surface glycoprotein without protease activity, although its function remains unknown at present.
Abstract: Dipeptidyl peptidase IV-related protein (DPPX) was found to be preferentially expressed in the brain tissue. We isolated two rat cDNA clones encoding DPPX-S and DPPX-L from a brain cDNA library, of which DPPX-L had a longer sequence at the NH2 terminus. The biosynthesis of DPPXs was examined in both in vitro and in vivo systems. In the cell-free translation system, DPPX-S and DPPX-L were synthesized as 93-kDa and 97-kDa forms, respectively, which are in good agreement with the molecular masses estimated from their primary structure. In COS-1 cells transfected with the cDNAs, DPPX-S and DPPX-L were initially synthesized as 113-kDa and 117-kDa forms, respectively, with high-mannose type oligosaccharides, which were then converted to 115-kDa and 120-kDa forms, mostly with the complex-type sugar chains. Immunofluorescence-microscopic observations confirmed that both DPPXs were expressed on the cell surface. DPPXs were found to have no enzyme activity of DPPIV, even when they were mutated to have the consensus active-site sequence Gly-X-Ser-X-Gly for serine proteases. Immunoblot analysis of samples prepared from various rat tissues demonstrated that DPPX-S, but not DPPX-L, was detectable only in the brain tissue. These results indicate that, of the two isoforms, DPPX-S is preferentially expressed in the brain tissue as the surface glycoprotein without protease activity, although its function remains unknown at present.
TL;DR: The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi, which fits into a slowtight binding mechanism with a low dissociation constant.
Abstract: The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.
TL;DR: The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications.
TL;DR: TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells, suggesting that P450nor and denitrification are distributed more universally among fungi than was previously thought.
Abstract: Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought.
TL;DR: Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.
TL;DR: Results suggested that PROM belongs to the glutamyl endopeptidase class, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.
TL;DR: Northern blot analysis of various human tissues revealed high levels of expression of the bile acid beta-glucosidase mRNA (3.6-kilobase message) in brain, heart, skeletal muscle, kidney, and placenta and lower levels ofexpression in the liver and other organs.
TL;DR: In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer and microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.
TL;DR: This is the first report on the primary structure of putative Na(+)-transporting ATPase from plant cells, cloned from marine alga Heterosigma akashiwo and shown around 40% identity in amino acids withNa(+)/K(+-ATPase alpha-subunits.
TL;DR: Sequence comparison with prevision studies showed that the distribution of charged and hydrophobic regions in equine κ-casein was similar, but not identical, to that found in the bovine protein; these regions are associated with the role of κ -casein in the formation and stabilization of the micellar structure of casein in milk.
Abstract: In this work the purification and the complete primary structure of κ-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' κ-casein and showed high similarity with κ-caseins from sources other than Equidae , confirming that this protein was indeed κ-casein in equine milk. The presence of post-translational modifications in equine κ-casein was investigated by mass spectroscopy, after enzymic dephosphorylation. Two main components were found, the smaller component being more abundant. Equine κ-casein was recognized by a lectin specific for one of the glucosidic bonds in the saccharide moiety of bovine κ-casein. Sequence comparison with prevision studies showed that the distribution of charged and hydrophobic regions in equine κ-casein was similar, but not identical, to that found in the bovine protein; these regions are associated with the role of κ-casein in the formation and stabilization of the micellar structure of casein in milk.
TL;DR: Sixteen different genes were cloned from the venomous glands of Centruroides sculpturatus Ewing using RNA extracted from scorpions collected in Tucson, Arizona, and several genes clearly show sequences that suggest post-transcriptional modifications, during the maturation process.
TL;DR: The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.
Abstract: The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS).
Saporin-S9 has 253 amino acids with a calculated molecular mass of 28 492.99, which is in good agreement with that determined by ESMS (28 495 ± 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27 789.49, in good agreement with that determined by ESMS (27 789 ± 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges. Copyright © 2001 John Wiley & Sons, Ltd.
Abbreviations:
ESMS
electrospray mass spectrometry
hsDNA
herring sperm DNA
CPase
carboxypeptidase
HSer
homoserine
LC
liquid chromatography
MAP
Mirabilis antiviral protein isolated from Mirabilis jalapa roots
MAP-S
RIP isolated from Mirabilis jalapa seeds
RIP
ribosome-inactivating protein
RP-HPLC
reversed-phase high-performance liquid chromatography
saporin-S
RIP isolated from Saponaria officinalis seeds
u
mass unit.
The standard one- or three-letter code has been used for the amino acids.
TL;DR: It is shown that substitution of alanine for Glu94, which occurs in the HEXXH zinc-binding motif of BMP-1, abolishes PCP activity, and mutation of residues Lys87 and Lys176 reduced the proteolytic activity of B MP-1 by ∼50%.
TL;DR: The purpose of this study was to synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum and confirm its chemical integrity by a variety of analytical techniques.
Abstract: The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high β-turn and α-helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.
TL;DR: It is suggested that, if sequential reactions are ordered throughout the Golgi stack, the complete O-glycosylation of the mucin molecules seems to be finely tuned to respond to specific damage to, or attack on, epithelia.
Abstract: The recombinant proteins of the two novel UDP-N-acetylgalactosamine (GalNAc) glycopeptide:N-acetylgalactosaminyltransferases (designated gpGaNTase-T7 and gpGaNTase-T9) were assayed with O-glycosylated products obtained from the prior action of the ubiquitous transferases (GaNTase-T1 and GaNTase-T2) towards MUC5AC mucin motif peptides (GTTPSPVPTTSTTSAP and peptides with single amino acid substitutions, GTTPSAVPTTSTTSVP and GTTPSPVPTTSITSVP, that are a reflection of mucin molecule polymorphism). gpGaNTase-T9 is known to be expressed differentially and more abundantly than gpGaNTase-T7 in some tissues; the results of in vitro glycosylation also indicates a difference in acceptor substrate specificities between the gpGaNTase isoforms. With the use of capillary electrophoresis, MS and Edman degradation, our study suggests that, in the O-glycosylation of mucin-type proteins, approach and recognition signalling by gpGaNTase-T7 and gpGaNTase-T9 depend largely on the peptide's primary structure (for example the presence of multiple clusters of hydroxy amino acids and the number of GalNAc residues attached to the peptide backbone). O-glycosylation in terms of sites of attachment seems to be less random than previously described and, if sequential reactions are ordered throughout the Golgi stack, the complete O-glycosylation of the mucin molecules seems to be finely tuned to respond to specific damage to, or attack on, epithelia.